Truncated forms of NY-ESO-1 protein

ABSTRACT

The invention relates to antibodies which bind to the cancer associated antigen NY-ESO-1. Both polyclonal and monoclonal antibodies are part of the invention, as are chimeric forms of the antibodies, and binding portions of antibodies. Uses of these antibodies are described. Also described are truncated, recombinant forms of the cancer associated antigen.

RELATED APPLICATION

This application is a Divisional Application of 09/062,422, filed Apr.17, 1998, now U.S. Pat. No 6,252,052 which is a continuation-in-part ofU.S. Ser. No. 08/937,263, filed Sep. 15, 1997, now U.S. Pat. No.6,274,145, which is a continuation-in-part of Ser. No. 08/725,182, filedOct. 3, 1996, now U.S. Pat. No. 5,804,381. Both of these applicationsare incorporated by reference.

FIELD OF THE INVENTION

This invention relates to antibodies against an antigen associated withcancer as well as the uses of these, as well as variants of thisprotein, and HLA binding peptides derived from it.

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets”, i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369: 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens is best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, etal., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer, associatedantigens.

One key methodology is described by Sahin, et al., Proc. Natl. Acad.Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, seeU.S. Pat. No. 5,698,396. Both of these references are incorporated byreference. To summarize, the method involves the expression of cDNAlibraries in a prokaryotic host. (The libraries are secured from a tumorsample). The expressed libraries are then immnoscreened with absorbedand diluted sera, in order to detect those antigens which elicit hightiter humoral responses. This methodology is known as the SEREX method(“Serological identification of antigens by Recombinant ExpressionCloning”). The methodology has been employed to confirm expression ofpreviously identified tumor associated antigens, as well as to detectnew ones. See the above referenced patent applications and Sahin, etal., supra, as well as Crew, et al., EMBO J 144: 2333-2340 (1995).

The SEREX methodology has been applied to esophageal cancer samples, andan antigen has now been identified, and its encoding nucleic acidmolecule isolated and cloned. This is the subject of the applicationswhich are incorporated by reference. The antigen and truncated formshave been found to be reactive with antibodies in the serum of cancerpatients. This, inter alia, is the subject of the invention, which isdescribed in more detail in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B, shows the expression pattern of RNA for the NY-ESO-1antigen, in various tissue types.

FIG. 2 shows Northern Blot analysis of NY-ESO-1 mRNA, which was found intestis and cell line SK-MEL-19, but not in various other cell and tissuesamples.

FIG. 3 shows potential sites for modification of the deduced amino acidsequence of NY-ESO-1.

FIG. 4 is a hydrophilicity plot of NY-ESO-1, showing hydrophilic domainsin the amino terminus and a long, hydrophobic stretch close to thecarboxyl end.

FIG. 5 shows the results of CTL lysis studies using various cells whichare HLA-A2 positive, NY-ESO-1 positive, positive for both, or positivefor neither.

FIG. 6 presents data establishing that HLA-A2 is the presenting moleculefor presentation of SEQ ID NO: 1 derived peptides.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Total RNA was extracted from a snap frozen specimen of well tomoderately differentiated squamous cell cancer of the esophagus, usingwell known methods. See, e.g., Chomzynski, J. Analyt. Biochem. 162:156-159 (1987), for one such method. This RNA was used to prepare a cDNAlibrary which was then transfected into λZAP phage vectors, inaccordance with the manufacturer's instructions. The λZAP library wasthen transfected into E. coli, yielding 1.6×10⁶ primary isolates.

The SEREX methodology of Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995), incorporated by reference, was then used. In brief,autologous serum was stripped of antibodies against molecules which areendogenous to E. coli by combining the serum with lysates of E. colitransfected with phage λZAP which did not contain the cDNA clones fromthe esophageal cancer cells.

The depleted serum was then diluted, and mixed with nitrocellulosemembranes containing phage plaques. The plaques were incubatedovernight, at room temperature. Washing followed, and then the filterswere incubated with alkaline phosphatase conjugated goat anti human FCYsecondary antibodies, and reactive phage plaques were visualized byincubating with 5-bromo-4-chloro-indolyl phosphate and nitrobluetetrazolium. A total of 13 positive clones were found.

EXAMPLE 2

Following identification, the reactive clones were subcloned tomonoclonality via dilution cloning and testing with human serum. Theseclones were then purified, excised in vitro, and converted into pBK-CMVplasmid forms using the manufacturer's instructions. The inserted DNAwas then evaluated using EcoRI-XbaI restriction mapping to determinedifferent inserts. Eight different inserts were identified, ranging insize from about 500 to about 1.3 kilobase pairs. The clones weresequenced using an ABI PRISM automated sequencer.

Table 1 summarizes the results. One gene was represented by fouroverlapping clones, a second by three overlapping clones, and theremaining six by one clone only.

A homology search revealed that the clones referred to as NY-ESO-2, 3,6, 7 were already known. See Elisei, et al., J. Endocrin. Invest. 16:533-540 (1993); Spritz, et al., Nucl. Acids Res. 15: 10373-10391 (1987);Rabbits, et al., Nature Genetics 4: 175-180 (1993); Crozat, et al.,Nature 363: 640-644 (1993); GenBank H18368 and D25606. Two of the clones(NY-ESO-3 and NY-ESO-6), have previously been shown to be expressed invarious normal human tissues. No evidence of lineage restriction hasbeen found. NY-ESO-6 (cDNA), appears to be the 3′-untranslated portionof the FUS/TLS gene. In experiments not reported here, sequencing andSouthern Blot analysis of NY-ESO-6 showed no evidence of translocationor point mutations in the cancer. Four of the clones, i.e., NY-ESO-1, 4,5 and 8 showed no strong homology to sequences in the databasesexamined, and were thus studied further.

TABLE 1 Genes isolated from esophageal cancer library by immunoscreeningwith autologous serum GENE CLONE# Size DNA databank Comments NY-ESO-1E1-5b 679 bp No strong expressed in testis E1-114b 614 bp homology andovary E1-153c 670 bp E1-50 679 bp NY-ESO-2 E1-71a 605 bp U1 small clonedby Ab E1-140 874 bp nuclear RNP 1 screening E1-31 750 bp homolog(thyroiditis patient) NY-ESO-3 E1-141b 517 bp Colon 3′ direct (dbjD25606, MboI cDNA; gb H18638) Adult brain unpublished cDNA NY-ESO-4E1A-10c 400 bp No strong ubiquitous homology expression in normaltissues NY-ESO-5 E1A-54 670 bp No strong expressed in normal homologyesophagus NY-ESO-6 E1B-9b −1.2 kb Human fus translocated in mRNAliposarcoma t (12; 16) NY-ESO-7 E1B-20f −1.0 kb human U1-70k differentfrom sn RNP NY-ESO-2 (embl HSU17052, gbM22636) NY-ESO-8 E1B-20g −1.3 kbNo strong ubiquitous homology expression in normal tissues

EXAMPLE 3

Studies were carried out to evaluate mRNA expression of the NY-ESO 1, 4,5 and 8 clones. To do this, specific oligonucleotide primers weredesigned for each sequence, such that cDNA segments of 300-400 basepairs could be amplified, and so that the primer

melting temperature would be in the range of 65-70° C. Reversetranscription-PCR was then carried out using commercially availablematerials and standard protocols. A variety of normal and tumor celltypes were tested. The clones NY-ESO-4 and NY-ESO-8 were ubiquitous, andwere not studied further. NY-ESO-5 showed high level expression in theoriginal tumor, and in normal esophageal tissue, suggesting that it wasa differentiation marker.

NY-ESO-1 was found to be expressed in tumor mRNA and in testis, but notnormal colon, kidney, liver or brain tissue. This pattern of expressionis consistent with other tumor rejection antigen precursors.

EXAMPLE 4

The RT-PCR assay set forth supra was carried out for NY-ESO-1 over amuch more complete set of normal and tumor tissues. Tables 2, 3 and 4show these results. In brief, NY-ESO-1 was found to be highly expressedin normal testis and ovary cells. Small amounts of RT-PCR productionwere found in normal uterine myometrium, and not endometrium, but thepositive showing was not consistent. Squamous epithelium of various celltypes, including normal esophagus and skin, were also negative.

When tumors of unrelated cell lineage were tested, 2 of 11 melanomascell lines showed strong expression, as did 16 of 67 melanoma specimens,6 of 33 breast cancer specimens and 4 of 4 bladder cancer. There wassporadic expression in other tumor types.

TABLE 2 mRNA distribution of NY-ESO-1 in normal tissues Tissue mRNATissue mRNA Esophagus − Adrenal − Brain* − Pancreas − Fetal Brain −Seminal Vesicle − Heart − Placenta − Lung − Thymus − Liver − Lymph node− Spleen − Tonsil − Kidney − PBL − Stomach − PBL, activated# − Smallintestine − Melanocytes − Colon − Thyroid − Rectum − Uterus +/−** Breast− Testis + Skin − Ovary + *tissues from several parts tested with IL-2and PHA **weakly positive in some specimens, negative by Northern blot

TABLE 3 mRNA distribution of NY-ESO-1 in melanoma and breast cancer celllines: Cell line NY-ESO-1 mRNA MZ2-MEL3.1 − MZ2-MEL2.2 − SK-MEL-13 −SK-MEL-19 + SK-MEL-23 − SK-MEL-29 − SK-MEL-30 − SK-MEL-31 − SK-MEL-33 −SK-MEL-37 + SK-MEL-179 − SK-BR-3 − SK-BR-5 − 734B − MDA-MB-231 −

TABLE 4 NY-ESO-1 mRNA expression in various human tumors by RT-PCR mRNAmRNA tumor type (positive/total) tumor type (positive/total) melanoma25/77  ovarian cancer 2/8 breast cancer 17/43  thyroid cancer 2/5prostate cancer 4/16 bladder cancer  9/13 colon cancer 0/16 Burkitt'slymphoma 1/2 glioma 0/15 basal cell carcinoma 0/2 gastric cancer 0/12Jejomyosarcoma 0/2 lung cancer 5/17 other sarcomas 0/2 renal cancer 0/10pancreatic cancer 0/2 lymphoma* 0/10 seminoma 0/1 hepatoma 2/7  spinalcord tumor 0/1 *non-Hodgkin's, non-Burkitt's types

A further set of experiments were carried out to ascertain if thepresence of anti NY-ESO-1 antibody in cancer patient sera could bedetermined via an ELISA.

To elaborate, recombinant NY-ESO-1 in a solution of coating buffer (15mM Na₂CO₃, 30 mM NaHCO₃, pH 9.6, 0.02% NaN₃), at a concentration of 1ug/ml, was adsorbed to microwell plates (10 ul of solution per well),and then kept overnight at 4° C. The plates were washed with phosphatebuffered saline, and blocked, overnight, at 4° C., with 10 ul/well of 2%bovine serum albumin/phosphate buffered saline. After washing, 10ul/well of diluted serum in 2% bovine serum albumin was added to thewells. Following two hours of incubation at room temperature, plateswere washed, and 10 ul/well of goat anti-human IgG-alkaline phosphataseconjugates were added, at a 1:1500 dilution. This solution was incubatedfor one hour at room temperature, followed by washing and addition of asolution of substrate for the alkaline phosphatase (10 ul/well). After25 minutes at room temperature, the wells were read with a fluorescenceplate reader. The results are presented in the following table:

Cancer patients: Eso 1 +/total tested % melanoma 12/127 9.4 ovariancancer 4/32 12.5 lung cancer 1/24 4.0 breast cancer 2/26 7.7 Blooddonors 0/70 0

In order to determine whether there was a relationship betweenexpression of mRNA for NY-ESO-1 in tumors, and antibody response to theNY-ESO-1 protein, data from sixty-two melanoma patients were compared.All patients whose serum was reactive with NY-ESO-1 protein (i.e.,contained antibodies to NY-ESO-1), also had NY-ESO-1 positive tumors,while no patients with NY-ESO-1 negative tumors showed antibodies toNY-ESO-1 in their serum. There was a percentage of NY-ESO-1 positivepatients who lacked the antibody. Given that about 20-40% of melanomasexpressed NY-ESO-1, and only patients with NY-ESO-1 positive tumors haveantibody, the data suggest a high percentage of patients with NY-ESO-1positive tumors develops antibodies against the protein, thus suggestinga broad scale assay useful in diagnosis and responsiveness to treatment.

EXAMPLE 5

Northern blot analysis was then carried out to investigate the size ofthe NY-ESO-1 transcript, and to confirm tissue expression patterns. Themethodology of Ausubel, et al., Current Protocols In Molecular Biology(John Wiley & Sons, 1995) was used. To be specific, 20 ug of total RNAper lane were dissolved in a formamide and formaldehyde containingbuffer; heated to 65° C., and then separated on a 1.2% agarose gel, with3% formaldehyde, followed by transfer to nitrocellulose paper.Hybridization was then carried out using a ³²P labelled probe, followedby high stringency washing. The final wash was at 0.1×SSC, 0.1% SDS, 60°C., for 15 minutes.

RNA from testis, and a melanoma cell line (SK-MEL-19) which had beenpositive for NY-ESO-1 in the prior assays, showed an RNA transcript ofabout 0.8-0.9 kb. An esophageal carcinoma specimen showed a smear in the0.4-0.9 kb range, reflecting partial degradation. RNA from additionaltissues or cell lines tested showed no transcript.

To get cDNA encoding the full transcript, the esophageal cDNA librarywas rescreened, using plaque hybridization, and the original cDNA cloneas the hybridization probe. When 3×10⁵ clones were screened, sixpositives were found. The three longest clones were sequenced. Analysisof open reading frames showed that all three contained the entire codingregion, and 5′-untranslated regions of variable size. The longest clone,752 base pairs in length, (excluding polyA), contains a 543 base paircoding region, together with 53 untranslated bases at the 5′ end and 159untranslated base pairs at the 3′-end. See SEQ ID NO: 1 (also, FIG. 3,SEQ ID NO: 8).

The long ORF indicated that the deduced sequence of NY-ESO-1 protein is180 amino acids. The single immunopositive clone contained a sequenceencoding 173 of these. Deduced molecular mass is 17,995 daltons.

Analysis shows that there is an abundance of glycine residues in theN-terminal portion (30 of the first 80, 4 in the remaining 100).Hydrophilicity analysis indicated that there were hydrophilic antigenicsequences in the N-terminal half of the molecule, with alternatinghydrophobic and hydrophilic sequences, ending with a long, C-terminalhydrophobic tail (amino acids 152-172), followed by a short hydrophilictail. This pattern suggests a transmembrane domain. There are severalpotential N-myristorylation sites, 3 phosphorylation sites, and noevidence of N-glycosylation sites

EXAMPLE 6

A melanoma cell line “NW-MEL-38” was established, in 1995, from apatient who suffered from malignant melanoma. Serum samples, peripheralblood lymphocytes and tumor samples, were taken from the subject andfrozen, until the work described herein was carried out. In anticipationof evaluating antitumor T cell response in this patient, the patient wasHLA typed as HLA-A1 and HLA-A2.

To determine whether melanoma from this patient expressed NY-ESO-1,total RNA was isolated from both tumor samples and cell line NW-MEL-38,using standard techniques. Then, two micrograms of the total RNA, fromeach samples were subjected to cDNA synthesis, again using standardtechniques.

The cDNA was then used in RT-PCR experiments, using the followingprimers:

(SEQ ID NO: 2), 5′-CACACAGGAT CCATGGATGC TGCAGATGCG G′-3′ and (SEQ IDNO: 3), CACACAAAGC TTGGCTTAGC GCCTCTGCCC TG-3′

These primers should amplify a segment of SEQ ID NO: 1 which spansnucleotides 271 to 599.

Amplification was carried out over 35 cycles, using an annealingtemperature of 60° C. The PCR products were visualized via ethidiumbromide staining, on a 1.5% agarose gel.

The results indicated that both the tumor and the cell line expressedSEQ ID NO: 1. The cell line and tumor samples were used in subsequentexperiments.

EXAMPLE 7

The isolated cDNA molecule, discussed supra, was then used to makerecombinant protein. Specifically, the cDNA was PCR amplified, usingstandard techniques, and was then cloned into a commercially availableplasmid vector, i.e., pQE9, which contains His tags. In work notelaborated upon herein, a second vector, pQE9K was also used. Thisdiffers from PQE9 in that kanamycin resistance is imparted by pQE9K,rather than ampicillin resistance.

The plasmid vector was transformed into E. coli strain XL1-Blue, andpositive transformants were identified via restriction mapping and DNAsequencing. Production of recombinant protein was induced usingisopropyl β-D-thiogalactoside, and the protein was purified on an Ni²⁺ion chromatography column, following well known procedures. The proteinwhen analyzed via 15% SDS-PAGE and silver staining, was identified as aprotein with a molecular weight of about 2 kilodaltons. This isconsistent with the anticipated size of the protein from its sequence.Two other forms of the recombinant protein were also identified. Theseconsisted of amino acids 10-180, and 10-121 of the amino acid sequencereported in SEQ ID NO: 8. They have molecular weights of about 20 kD and14 kD, respectively, on SDS-PAGE, as carried out supra.

An additional set of experiments were carried out to express NY-ESO-1 inbaculovirus. To elaborate, the NY-ESO-1 cDNA insert was released fromthe pQE9 vector, by cleavage with BamHI and HindIII. This insert wasthen subcloned into a commercially available baculovirus vector whichhad been cleaved with the same enzymes. Positive clones were determined,using standard methods, and transfected into recipient Sf9 cells.Recombinant viruses were then used to infect insect cells, using astandard medium (IPL-41), supplemented with 10% fetal calf serum. Themultiplicity of infection for the work was 20. Expression of recombinantprotein was determined as described supra. The recombinant proteinproduced in this vector carries an His-tag, so it was purified on Ni²⁺affinity columns, also as described, supra. The protein consists ofamino acids 10-180, and has a molecular weight of 20 kD via SDS-PAGE.

Additional eukaryotic transfectants were then produced. To do this, theNY-ESO-1 coding sequence was isolated from the pQE9 vector describedsupra, and then cloned into BamHI-HindIII sites of eukaryotic expressionvector pcDNA 3.1. Next, COS-7 cells were transfected with this vector,by contacting cell samples with 150 ng of the plasmid discussed supra,and 150 ng of plasmid pcDNA 1 Amp, which contained either cDNA forHLA-A2.1 or cDNA for HLA-A1, The well known DEAE-dextran chloroquinemethod was used. The cells were then incubated at 37° C., for 48 hours,after which they were tested in a CTL stimulation assay. Specifically,the assay followed Traversari et al, Immunogenetics 35: 145-148 (1992),incorporated by reference. In brief, 2500 CTLs, (NW38-IVS-1, see example9, infra), in 100 ul RPMI supplemented with 100% human serum, and 25U/ml of recombinant IL-2 were added to microwells containing COS-7transfectants (20,000 cells/well) After 24 hours, 50 ul of supernatantwere collected from each well, and TNF-α levels were determined in astandard assay, i.e., one where cytotoxicity against WEHI 164 clone 13cells were tested, using MTT. Positive cells were used in the WesternBlot analysis, described in the example which follows.

The CTLs used were CTL NW38-IVS-1, prepared in accordance with Knuth etal., Proc. Natl. Acad. Sci. USA 81: 3511-3515 (1984), incorporated byreference. Specifically, mixed lymphocyte T cell cultures were set up,by combining 10⁵ autologous NW38 MEL-1 tumor cells, and 10⁶ peripheralblood lymphocytes, taken from the subject. The cytokine IL-2 was added,and the mixed culture was incubated for one week at 37° C. Tumor cellswere removed, and a new aliquot of 5×10⁴ tumor cells were added togetherwith IL-2. This process was repeated weekly, until a strong response wasseen when tested against ⁵¹Cr labelled NW-MEL-38 cells. The responder Tcells were collected and frozen until used in further experiments.

EXAMPLE 8

Western Blot analysis was then carried out, using the serum samplesdescribed supra, as well as cell lysates taken from the cell lineNW-MEL-38, described supra, and the COS-7 transfectants, describedsupra, and the purified recombinant protein, also described supra. Serumsamples were taken from various points of the patient's therapy. Therewas no difference in the results.

In these assays, 1 ug of recombinant NY-ESO-1 protein, or 5 ul of celllysates of either type were diluted in SDS and boiled for five minutes,and then electrophoresed on a 15% SDS gel. After overnight blotting onnitrocellulose (0.45 um), and blocking with 3% BSA, the blots wereincubated with serum, diluted at 1:1000, 1:10,000, and 1:100,000, orwith a monoclonal antibody against NY-ESO-1, diluted to 1:50, as apositive control. The monoclonal antibody was prepared via Chen, et al.,Proc. Natl. Acad. Sci. USA 5915-5919 (1996), incorporated by referenceand elaborated as follows. BALB/C mice were immunized via fivesubcutaneous injections of recombinant NY-ESO-1 protein, at 2-3 weekintervals. The immunizing formulation included 50 ug of recombinantprotein in adjuvant. The first injection used Complete Freund'sAdjuvant, and Incomplete Freund's Adjuvant was used thereafter. Spleencells were taken from the immunized mice, and fused with mouse myelomacell line SP2/0, to generate hybridomas. Representative hybridoma E978was used for generation of mAbs.

Once hybridomas were generated, they were cloned, and their supernatantswere screened against recombinant protein, using a standard solid phaseELISA on microtiter plates. The assay was in accordance with Dippold etal., Proc. Natl. Acad. Sci. USA 77: 6114-6118 (1980), incorporated byreference. A series of negative controls were also run, usingrecombinant NY-ESO-1. Serum antibodies which bound to recombinantprotein, produced by E. coli as described, supra were visualized usinggoat anti-human IgG, labelled with alkaline phosphatase at 1:10,000dilution, and were then visualized with NBT-phosphate. UntransfectedCOS-7 cells were also used as a control. Serum from a healthy individualwas also used as a control.

Strong reactivity against the recombinant protein was found at serumdilutions down to 1:100,000, and there was also reactivity againstlysate of NW-MEL-38. There was no reactivity found against theuntransfected COS-7 cells, nor did the serum from a healthy individualshow reactivity.

EXAMPLE 9

Four different forms of NY-ESO-1 are described supra, i.e., the formproduced by SEQ ID NO: 1 in E. coli, as well as one consisting of aminoacids 10-180, one consisting of amino acids 10-121, and a form,expressed in the baculovirus vector system discussed supra whichconsisted of amino acids 10-180. Each form was used in ELISAs, followingthe above described protocols. All forms of the protein were found to beequally reactive with antibodies taken from various patients, as well asthe murine monoclonal antibodies discussed, supra.

EXAMPLE 10

In the testing of the COS-7 transfectants, supra, and the assaysdiscussed in this example, a cytolytic T cell line “NW38-IVS-1” wasused. This “CTL” was generated, via in vitro stimulation of theperipheral blood lymphocytes mentioned supra, using the tumor cell lineNW-MEL-38. This was done using standard techniques.

The CTL was used in a cytotoxicity assay with NW-MEL-38 (which wasHLA-A1, A2 positive, and NY-ESO-1 positive), along with two allogeneiccell lines which were NY-ESO-1 and HLA-A2 positive (SK-MEL-37 andMZ-MEL-19), a cell line which is MHC Class I negative (SK-MEL-19), acell line which is HLA-A2 positive, but NY-ESO-1 negative (NW-MEL-145),along with control cell lines K562 and autologous phytohemagglutininstimulated blasts. Various effector/target ratios were used, and lysisof ⁵¹Cr labelled target cells was the parameter measured. FIG. 5 showsthis.

The results indicated that the CTL NW38-IVS-1 lysed both the autologouscell line NW MEL-38, and the allogeneic cell lines which were HLA-A2 andESO-1 positive. Hence, the CTL was reactive with allogeneic materials.See FIG. 6.

EXAMPLE 11

As patient NW38 was HLA-A1 and HLA-A2 positive, experiments were carriedout to determine which MHC molecule was the presenting molecule.

The same experiment, described supra with COS-7 cells was carried out,except that, in these experiments, care was taken to secure separategroups of cotransformants which had been transformed with either HLA-A1cDNA, or HLA-A2 cDNA, but not both. These results show that the CTLNW38-IVS-1 lysed COS-7 transfectants containing both NY-ESO-1 and HLA-A2exclusively. See FIG. 6. The work also confirmed the specificity of theCTL, since the NY-ESO-1 negative, HLA-A2 positive cells described inExample 9 were positive for other molecules known to be processed topeptides presented by HLA-A2 molecules.

EXAMPLE 12

Once the presenting MHC molecule was identified as HLA-A2, a screeningof the amino acid sequence for NY-ESO-1 was carried out, to identify allpeptides which satisfy this motif, using the model set forth by D'Amaroet al., Human Immunol. 43: 13-18 (1995), and Drijfhout, et al., HumanImmunol. 43: 1-12 (1995) incorporated by reference. Peptidescorresponding to all of the amino acid sequences deduced thereby weresynthesized, using standard techniques, and were then used incytotoxicity assays, following Knuth et al., Proc. Natl. Acad. Sci. USA81: 3511-3515 (1984), incorporated by reference. Specifically, cell lineCEMX721.174.T2 (“T2” hereafter), was used, because it does not processantigens to MHC complexed peptides, thereby making it ideal forexperiments of the type described herein. Samples of T2 cells werelabelled with 100 uCi of Na(⁵¹Cr)O₄, using standard methods, and werethen washed three times, followed by incubation with 10 ug/ml peptideand 2.5 ug/ml of β2-microglobulin. Incubation was for one hour, at roomtemperature. Then responder cells (100 ul of a suspension of CTLNW38-IVS-1) were added, at an effector/target ratio of 90:1, andincubated for four hours in a water saturated atmosphere, with 5% CO₂,at 37° C. Then, plates were centrifuged at 200×g for five minutes, 100ul of supernatant was removed, and radioactivity was measured. Thepercentage of ⁵¹Cr release was determined in accordance with knownstrategies. it was found that the peptides SLLMWITQCFL (SEQ ID NO: 4),SLLMWITQC (SEQ ID NO: 5), and QLSLLMWIT (SEQ ID NO: 6), were the threebest stimulators of CTLs. Comparable results were found when NW-MEL-38and cell lines SK-MEL-37 and MZ-MEL-19 were used as targets, as isshown, supra.

EXAMPLE 13

The amino acid sequence of the protein encoded by SEQ ID NO: 1 wasanalyzed for peptide sequences which correspond to HLA binding motifs.This was done using the algorithm taught by Parker et al., J. Immunol.142: 163 (1994), incorporated by reference. In the Table which follows,the amino acid sequence, the HLA molecule to which it presumably binds,and the positions in SEQ ID NO: 1 are given. The resulting complexesshould provoke a cytolytic T cell response. This could be determined byone skilled in the art following methods taught by, e.g., van derBruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994), incorporatedby reference.

MHC/HLA Sequence Molecule Positions GPESRLLEF HLA-A1 82-90 LLMWTTQCFHLA-A3 158-166 LMWITQCFL HLA-A3 159-167 EPTVSGNIL HLA-A24 125-133LQLSISACL HLA-A24 145-153 GARGPESRL HLA-B7 79-87 APRGPHGGA HLA-B7 60-68ESRLLEFYL HLA-B7 84-92 APPLPVPGV HLA-B7 113-121 FATPMEAEL HLA-B7  96-104AADHRQLQL HLA-B7 139-147 GARGPESRL HLA-B8 79-87 ESRLLEFYL HLA-B8 84-92VPGVLLKEF HLA-B35 118-126 ESRLLEFYL HLA-B35 84-92 GARGPESRL HLA-B3579-87 LEFYLAMPF HLA-B44 88-96 PESRLLEFY HLA-B44 83-91 AELARRSLA HLA-B44102-110 MEAELARRS HLA-B44 100-108 QQLSLLMWI HLA-B52 154-162 AQDAPPLPVHLA-B52 110-118 LQLSISSCL HLA-B52 145-153 ITQCFLPVF HLA-B52 162-170LLEPYLAMPF HLA-A1 87-96 GPESRLLEFY HLA-A1 82-91 PLPVPGVLLK HLA-A3115-124 RSLAQDAPPL HLA-A24 107-116 APPLIPVPGVL HLA-B7 113-122 GARGPESRLLHLA-B7 79-88 GPHGGAASLG HLA-B7 63-72 APRGPHGGAA HLA-B7 60-69 GPRGAGAARAHLA-B7 44-53 TAADHRQLQL HLA-B8 138-147 APPLPVPGVL HLA-B52 113-122QQLSLLMWIT HLA-B52 154-163 LQQLSLLMWI HLA-B52 153-162 KEFTVSFNIL HLA-B52124-133

The foregoing examples describe the isolation of a nucleic acid moleculewhich encodes an esophageal cancer associated antigen. “Associated” isused herein because while it is clear that the relevant molecule wasexpressed by esophageal cancer, other cancers, such as melanoma, breast,prostate and lung also express the antigen.

The invention relates to those nucleic acid molecules which encodeantigens as described, and which hybridize to reference sequence SEQ IDNO: 1 under stringent conditions. “Stringent conditions” as used hereinrefers to conditions such as those specified in U.S. Pat. No. 5,342,774,i.e., 18 hours of hybridization at 65° C., followed by four one hourwashes at 2×SSC, 0.1% SDS, and a final wash at 0.2×SSC, more preferably0.1×SSC, 0.1% SDS for 30 minutes, as well as alternate conditions whichafford the same level of stringency, and more stringent conditions.

Also a part of the invention are expression vectors which incorporatethe nucleic acid molecules of the invention, in operable linkage (i.e.,“operably linked”) to a promoter. Construction of such vectors is wellwithin the skill of the art, as is the transformation or transfection ofcells, to produce eukaryotic cell lines, or prokaryotic cell strainswhich encode the molecule of interest. Exemplary of the host cells whichcan be employed in this fashion are COS cells, CHO cells, yeast cells,insect cells (e.g., Spodoptera frugiperda), NIH 3T3 cells, and so forth.Prokaryotic cells, such as E. coli and other bacteria may also be used.

Also a part of the invention is the antigen described herein, both inoriginal peptide form and in post translational modified form, as wellas proteins consisting of at least amino acids 10-121, and no more than10-180, of the protein encoded by SEQ ID NO: 1. The molecule is largeenough to be antigenic without any posttranslational modification, andhence it is useful as an immunogen, when combined with an adjuvant (orwithout it), in both precursor and post-translationally modified forms.These proteins can be used to determine whether or not antibodies arepresent in a sample, such as serum or blood, as shown supra. Theantibodies produced using this antigen, both poly and monoclonal, arealso a part of the invention as well as hybridomas which make themonoclonal antibody. These can be used therapeutically or diagnosticallyas the whole molecule or in portions, as discussed infra. Also a part ofthe invention are reactive fragments, such as Fab, F(ab)₂′ and otherfragments, as well as chimeras, humanized antibodies, recombinantlyproduced antibodies, and so forth. Especially preferred are chimeraswhere the entire antibody but the complementarity determining regions“CDRS” is human, but the CDRs are murine.

As is clear from the disclosure, one may use the proteins and nucleicacid molecules of the invention diagnostically. The SEREX methodologydiscussed herein is premised on an immune response to a pathologyassociated antigen. Hence, one may assay for the relevant pathology via,e.g., testing a body fluid sample of a subject, such as serum, forreactivity with the antigen per se. Reactivity would be deemedindicative of possible presence of the pathology so, too, could oneassay for the expression of the antigen via any of the standard nucleicacid hybridization assays which are well known to the art, and need notbe elaborated upon herein. One could assay for antibodies against thesubject molecule, using standard immunoassays as well.

Analysis of SEQ ID NO: 1 will show that there are 5′ and 3′ non codingregions presented therein. The invention relates to those isolatednucleic acid molecules which contain at least the coding segment, i.e.,nucleotides 54-593, and which may contain any or all of nucleotides 1-53and/or 594-747 of SEQ ID NO: 1.

Further analysis, as discussed supra, reveals that the molecule isprocessed to peptides which provoke lysis by cytolytic T cells. Example7 showed how this type of motif analysis can be carried out for HLA-A2molecules. There has been a great deal of work in motifs for various MHCor HLA molecules, which is applicable here. Hence, a further aspect ofthe invention is a therapeutic method, wherein one or more peptideswhich bind to an HLA molecule on the surface of a patient's tumor cellsare administered to the patient, in an amount sufficient for thepeptides to bind to the MHC/HLA molecules, and provoke lysis by T cells.The exemplification given supra for HLA-A2 molecules is by no means theonly type of this administration that can be used. Any combination ofpeptides may be used, such as those for other HLA moleucles, describedsupra. These peptides, which may be used alone or in combination, aswell as the entire protein or immunoreactive portions thereof, may beadministered to a subject in need thereof, using any of the standardtypes of administration, such as intravenous, intradermal, subcutaneous,oral, rectal, and transdermal administration. Standard pharmaceuticalcarriers, adjuvants, such as saponins, GM-CSF, and interleukins and soforth may also be used. Further, these peptides and proteins may beformulated into vaccines with the listed material, as may dendriticcells, or other cells which present relevant MHC/peptide complexes.These peptides may also be used to form multimeric complexes ofHLA/peptides, such as those described by Dunbar, et al., Curr. Biol. 8:413-416 (1998), incorporated by reference, wherein fourpeptide/MHC/biotin complexes are attached to a streptavidin or avidinmolecule. Such complexes can be used to identify and/or to stimulate Tcell precursors.

Similarly, the invention contemplates therapies wherein the nucleic acidmolecule which encodes NY-ESO-1 is incorporated into a vector, such asan adenovirus based vector, to render it transfectable into eukaryoticcells, such as human cells. Similarly, nucleic acid molecules whichencode one or more of the peptides may be incorporated into thesevectors, which are then the major constituent of nucleic acid basestherapies.

Any of these assays can also be used in progression/regression studies.One can monitor the course of abnormality involving expression ofNY-ESO-1, simply by monitoring levels of the protein, its expression,and so forth using any or all of the methods set forth supra.

It should be clear that these methodologies may also be used to trackthe efficacy of a therapeutic regime. Essentially, one can take abaseline value for the NY-ESO-1 protein, using any of the assaysdiscussed supra, administer a given therapeutic agent, and then monitorlevels of the protein thereafter, observing changes in ESO-1 levels asindicia of the efficacy of the regime.

As was indicated supra, the invention involves, inter alia, therecognition of an “integrated” immune response to the NY-ESO molecule.One ramification of this is the ability to monitor the course of cancertherapy. In this method, which is a part of the invention, a subject inneed of the therapy receives a vaccination of a type described herein.Such a vaccination results, e.g., in a T cell response against cellspresenting HLA/peptide complexes on their cells. The response alsoincludes an antibody response, possibly a result of the release ofantibody provoking proteins via the lysis of cells by the T cells.Hence, one can monitor the effect of a vaccine, by monitoring an immuneresponse. As is indicated, supra, an increase in antibody titer or Tcell count may be taken as an indicia of progress with a vaccine, andvice versa. Hence, a further aspect of the invention is a method formonitoring efficacy of a vaccine, following administration thereof, bydetermining levels of antibodies in the subject which are specific forthe vaccine itself, or a large molecules of which the vaccine is a part.

The effects of a vaccine can also be measured by monitoring the Tcell-response of the subject receiving the vaccine. A number of assayscan be used to measure the precursor frequency of these in vitrostimulated T cells. These include, but are not limited to, chromiumrelease assays, TNF release assays, IFNγ release assays, an ELISPOTassay, and so forth. Changes in precursor T cell frequences can bemeasured and correlated to the efficacy of the vaccine. Additionalmethods which can be employed include the use of multimeric complexes ofMHC/peptides. An example of such complexes is the tetramericHLA/peptide-biotin-streptavidin system of Dunbar, et al. Curr. Biol. 8:413-416 (1998), incorporated by reference.

The identification of NY-ESO-1 proteins as being implicated inpathological conditions such as cancer also suggests a number oftherapeutic approaches in addition to those discussed supra. Theexperiments set forth supra establish that antibodies are produced inresponse to expression of the protein. Hence, a further embodiment ofthe invention is the treatment of conditions which are characterized byaberrant or abnormal levels of NY-ESO-1 proteins, via administration ofantibodies, such as humanized antibodies, antibody fragments, and soforth. These may be tagged or labelled with appropriate cystostatic orcytotoxic reagents.

T cells may also be administered. It is to be noted that the T cells maybe elicited in vitro using immune responsive cells such as dendriticcells, lymphocytes, or any other immune responsive cells, and thenreperfused into the subject being treated.

Note that the generation of T cells and/or antibodies can also beaccomplished by administering cells, preferably treated to be renderednon-proliferative, which present relevant T cell or B cell epitopes forresponse, such as the epitopes discussed supra.

The therapeutic approaches may also include antisense therapies, whereinan antisense molecule, preferably from 10 to 100 nucleotides in length,is administered to the subject either “neat” or in a carrier, such as aliposome, to facilitate incorporation into a cell, followed byinhibition of expression of the protein. Such antisense sequences mayalso be incorporated into appropriate vaccines, such as in viral vectors(e.g., Vaccinia), bacterial constructs, such as variants of the knownBCG vaccine, and so forth.

Also a part of the inventions are peptides, which can be nonamers,decamers, or undecamers defined as having a core sequence:

LLMWIT (SEQ ID NO: 7)

which have at least one additional residue terminal to the first Lresidue, preferably serine and may have as many as three, wherein Serineis linked to L to form -SL-, and 0-4 additional amino acids at theC-terminus which, as shown supra, bind to HLA-A2 molecules, therebyprovoking a CTL response. These peptides may be used therapeutically,via administration to a patient who is HLA-A2 positive, and expressesNY-ESO-1 in connection with a pathology, as well as diagnostically,i.e., to determine if HLA-A2 positive cells are present, or if relevantCTLs are present, and so forth.

The HLA-A2 molecule is an MHC Class I molecule, and T cells whichrespond to complexes of peptides and class I molecules are generallyCD8⁺ cells. Another subset of T cells, CD4⁺ cells, responds to complexesof MHC-Class II molecules and peptides, and MHC-Class II restricted CD4⁺T cell responses against recombinant NY-ESO-1, presented by autologouscultured dendritic cells have been detected in melanoma patients.Specifically, in results not described herein, CD4⁺ cells were separatedfrom other cells from PBLs or serum samples, using well knowntechniques. Then, they were admixed with dendritic cells which had beenpulsed with NY-ESO-1 protein. Proliferation of CD4⁺ cells was observed,bringing another facet to the integrated immune response discussedherein. Hence, a further aspect of this invention are these CD4⁺ Tcells, peptides which bind to the MHC-Class II molecules, and their usein therapy.

Other features and applications of the invention will be clear to theskilled artisan, and need not be set forth herein.

The terms and expression which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 752 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gc#catgcagg     60ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga gg#ccctggca    120ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc ac#gggcggca    180gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gc#cccgcggg    240gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gc#cagggggc    300cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc at#ggaagcag    360agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca gg#ggtgcttc    420tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gc#agaccacc    480gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg at#gtggatca    540cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cg#ctaagccc    600agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc ag#cacgagtg    660gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt ac#atgtttgt    720 ttctgtagaa aataaaactg agctacgaaa aa       #                   #         752 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:CACACAGGAT CCATGGATGC TGCAGATGCG G         #                  #          31 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 32 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:CACACAAAGC TTGGCTTAGC GCCTCTGCCC TG        #                  #          32 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 11 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Le #u                  5 #                   #10 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 9 amino  #acids          (B) TYPE: amino acid           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:Ser Leu Leu Met Trp Ile Thr Gln Cys                  5(2) INFORMATION FOR SEQ ID NO: 6:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 9 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:Gln Leu Ser Leu Leu Met Trp Ile Thr                  5(2) INFORMATION FOR SEQ ID NO: 7:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 6amino acid #s           (B) TYPE: amino acid          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7: Leu Leu Met Trp Ile Thr                 5 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 180          (B) TYPE: amino acid           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Se #r Thr Gly Asp Ala Asp                5   #                 10  #                 15Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pr #o Gly Gly Asn Ala Gly            20       #            25       #            30Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Ar #g Ala Pro Arg Gly Ala        35           #        40           #        45Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gl #y Ala Pro Arg Gly Pro    50               #    55               #    60His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cy #s Cys Arg Cys Gly Ala65                   #70                   #75                   #80Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Ty #r Leu Ala Met Pro Phe                85   #                90   #                95Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Ar #g Ser Leu Ala Gln Asp            100       #           105       #           110Ala Pro Pro Leu Pro Val Pro Gly Val Leu Le #u Lys Glu Phe Thr Val        115           #       120           #       125Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Al #a Ala Asp His Arg Gln    130               #   135               #   140Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gl #n Leu Ser Leu Leu Met145                 1 #50                 1 #55                 1 #60Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Le #u Ala Gln Pro Pro Ser                165   #               170   #               175Gly Gln Arg Arg             180

We claim:
 1. Isolated protein consisting of at least amino acids 10-121and no more than amino acids 10-180 of the protein encoded by theisolated nucleic acid molecule having the nucleotide sequence set forthin SEQ ID NO:
 1. 2. The isolated protein of claim 1, consisting of aminoacids 10-121.
 3. The isolated protein of claim 1, consisting of aminoacids 10-180.
 4. Immogenic composition comprising the isolated proteinof claim 1, and a carrier.
 5. An isolated tumor rejection antigenprecursor protein which is expressed by melanoma cells, wherein saidtumor rejection antigen precursor protein which elicits a humoralresponse by a human against said tumor rejection antigen precursorprotein is encoded by a cDNA molecule, the complementary sequence ofwhich hybridizes to the cDNA molecule consisting of nucleotides 54-600of SEQ ID NO: 1, at 65°C., 2×SSC, 0.1% SDS, followed by a wash at0.2×SSC, 0.1×SDS, for 30 minutes at 65°C.